There is a continuous need in medical practice, research and diagnostic procedures for rapid and accurate determinations of biological substances which are believed or known to be present in biological fluids. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, proteins, polysaccharides, prostaglandins or infectious organisms in blood, saliva, semen, vaginal secretions, urine, tissue cultures and other biological fluids and specimens has to be determined in a rapid and efficient manner for suitable diagnosis and treatment at a cost that is not prohibitive.
To provide such determinations, various methods have been devised for isolating and detecting the presence of a biological or chemical substance which participates in an immunological reaction, such as an antibody or antigenic material, drug or hapten. Such substances are identified herein as "immunoreactants", and biological or chemical substances which specifically bind (or react) with them to form an immunological complex are identified herein as "receptors". Often, one or the other reactants in such a complex, or even a third substance reactive with either of the first two reactants, is labeled with a detectable marker to provide a means for detecting the complex. Radioisotopes, enzymes, chemiluminescent moieties, fluorescent moieties and dyes have been generally used for this purpose.
Immunoassays have been growing in importance in recent years as a means for detecting the presence or amount of infectious agents in animals or humans. Generally, the infectious agent is detected by determining the presence or amount of an immunological complex formed from an antigenic component extracted therefrom, or by determining the presence or amount of antibodies to the infectious agent in a specimen.
In recent years, the use of enzyme labels has received increasing attention because they provide distinct advantages over other labels, particularly radioisotopes and fluorescent markers. Enzyme labels are becoming prominently used in immunoassays for detecting infectious agents in what are known in the art as competitive enzyme immunoassays (EIA), both direct and indirect enzyme linked immunosorbent assays (ELISA), and immunometric or "sandwich" assays.
One such infectious agent which can be detected by immunoassays is Chlamydia trachomatis (herein C. trachomatis) which is one of two microbial species of the genus Chlamydiaceae, order Chlamydiales. There are 15 or more strains of this species which are the causes of a number of human ocular and genital diseases including trachoma, inclusion conjunctivitis, lymphogranuloma venereum, nongonococcal urethritis and proctitis. Infection from C. trachomatis is pervasive in the general population so that it is believed that there are millions of cases each year of nongonococcal urethritis alone.
Gonorrhea is another disease usually transmitted by sexual contact, and is caused by a bacterium of the Neisseria genus, especially N. gonorrhoeae. The disease has plagued mankind for thousands of years, and although antibiotics have helped control its spread, it still persists in epidemic proportions in many parts of the world. The importance of detection and treatment of this organism is well recognized. N. meningitidis and N. lactamica are also species of considerable medical and diagnostic interest.
Because of the widespread nature of these diseases, there is considerable interest in having a rapid, simple and reliable test for detection of chlamydial and gonococcal organisms. Considerable research has been carried out to find useful ways to extract and detect antigens from chlamydial and gonococcal organisms.
Assays for C. trachomatis and N. gonorrhoeae carried out using a solid support are described in U.S. Pat. No. 4,497,899 and U.S. Pat. No. 4,497,900, respectively (both issued Feb. 5, 1985 to Armstrong et al and Abram et al, respectively). The described assays are performed by extracting antigen from the organism and coating it on a bare solid support. The coated antigen is then detected with either one or two antibodies, one of which is suitably labeled with an enzyme. The critical feature of the assays appears to be the use of a solid support for attachment which is untreated or uncoated with any biological material. Attachment of antigen is apparently achieved by incubating the coated support for an extended time sufficient to cause adsorption of antigen thereon. The absorption time is at least 30 minutes at elevated temperature (37.degree. C.). The entire assay described in U.S. Pat. No. 4,497,899 takes at least 3 hours to perform. A similar quicker assay is described in U.S. Pat. No. 4,497,900 for N. gonorrhoeae.
It would be desirable to have a much more rapid test for chlamydial or gonococcal organisms which has high reliability and can be performed at room temperature.
Such an improvement is described and claimed in copending U.S. Ser. No. 255,923 (filed Oct. 7, 1988 by Pronovost) now U.S. Pat. No. 5,075,220 (issued Dec. 24, 1991). It was found that ionically charged supports attract chlamydial or gonococcal antigen and enable one to quickly and sensitively detect such antigens. However, further improvements were needed for some biological specimens, especially those containing copious amounts of whole blood, mucus or components. Thus, the improvement described in copending U.S. Ser. No. 255,920 (filed Oct. 7, 1988 by Mauck) now U.S. Pat. No. 5,032,504 (issued Jul. 16, 1991) was made.
Despite the considerable improvements described in the copending applications noted above, there is a continued need to make the assay faster (that is, less than 20 minutes). It would be desirable to eliminate as many steps as possible from the earlier assay protocols so users would find the assays more convenient and suitable for prompt diagnosis and treatment of chlamydial and gonococcal infections. Moreover, the peroxidase-labeled antibody conjugate used in the known assays has a certain degree of stability (and thus, shelf life), but there is a need for additional stability and longer shelf life.
A highly rapid and accurate assay is described in more detail in copending U.S. Ser. No. 522,444 (filed on even date herewith by Mauck, Boyer, Warren III, Sprague and Snodgrass) and entitled "Use of Enzyme-Labeled Antibody Fragment in Determination of Chlamydial or Gonococcal Antigens". In this assay, increased rapidity of the assay is achieved because fewer steps are employed to detect the antigen. Moreover, an enzyme-labeled antibody F(ab').sub.2 fragment was found to be more stable than an enzyme-labeled whole antibody.
However, in developing that assay, which solved a number of problems with known assays, it was found that premature reaction of the enzyme-label (particularly, peroxidase) conjugated to antibody fragments produced unwanted background which obscured true positive signals in assays. This was particularly found to be a problem with the negative controls which were used to indicate whether the assay protocol was properly carried out. Although it is presently uncertain, it is speculated that the enzyme label on the antibodies may be involved in a reaction with residual substrate (that is, hydrogen peroxide when the enzyme is peroxidase) and phenolic electron transfer agents (such as 4'-hydroxyacetanilide) which are often used to enhance the rate of dye formation in the presence of the enzyme (see for example U.S. Pat. No. 4,828,983, issued May 9, 1989 to McClune). The result is unwanted dye formation.
Stable compositions of enzyme-labeled immunoreactants such as antibodies or antibody fragments are needed to provide rapid assays without undesirable background signal.